supplementary figure s1

RINDLD1 10.0HEK293 10.0HeLa 10.0MCF7 7.7Adipose 8.2Lung 9.0Ovary 8.7Brain 8.7Breast 4.3Colon 7.8Heart 9.6Kidney 8.8Liver 9.4Lymph Node 8.5Prostate 6.0Skeletal Muscle 9.9Testis 9.3Thyroid 8.9

A

Supplementary Figure S1

B


Number of library

Number of tags generated

Number of tags mapped to

upstream or first exon (%)

Number of tags mapped to

downstream or last exon (%)

Number of represented

NM genes by > 5ppm tags

Number of represented NR genes by > 5ppm tags

Number of genes

containing multiple TSCs

Number of genes

containing multiple PACs

DLD1 - 8,097,384 95% 74% 7,540 80 243 1,440

HEK293 - 3,367,796 95% 67% 7,768 113 227 1,622

HeLa - 5,635,548 95% 67% 7,488 91 323 1,550

MCF7 - 5,617,408 94% 64% 7,472 128 396 1,522

Adipose - 2,414,959 90% 77% 6,076 88 212 1,071

Lung - 2,533,045 84% 69% 4,545 52 201 759

Ovary - 3,921,177 93% 81% 5,138 90 247 790

Brain - 4,068,184 92% 85% 6,440 132 286 948

Breast - 3,154,960 94% 82% 6,620 101 312 1,145

Colon - 4,473,789 93% 85% 6,956 88 344 1,029

Heart - 4,513,913 94% 87% 4,026 56 205 614

Kidney - 3,378,219 94% 87% 6,074 115 272 948

Liver - 2,552,292 94% 89% 3,379 36 166 442

Lymph Node - 3,074,947 94% 88% 4,778 85 247 707

Prostate - 387,113 92% 72% 2,541 43 83 425

Skeletal Muscle - 2,430,014 93% 88% 4,647 74 268 690

Testis - 6,292,637 84% 85% 7,045 384 532 989

Thyroid - 6,382,769 94% 82% 6,626 106 312 1,155

Average - 4,016,453 92% 79% 5,983 107 281 1,033

Total 18 72,296,154 91% 76% 10,038 574 2,488 5,096

C


Number of library

Number of tags generated

Number of tags mapped to upstream

or first exon (%)

Number of represented

NM covered by >90%

Number of represented NM with all of the exons

covered by the tag

Number of represented NR

covered by > 90%

Number of representedNR with all of the exons

covered by the tag

DLD1 0.512

7,374,6995,731,4686,948,401

92% 425 1,053 87 313

HEK293 0.512

3,976,0254,094,1378,511,193

94% 335 1,430 72 353

HeLa 0.512

14,180,2625,284,0078,639,636

93% 442 2,040 67 345

MCF7 0.512

7,275,82612,813,3155,259,460

93% 338 1,728 73 357

Adipose 0.512

4,460,0473,723,2085,081,920

87% 593 1,768 48 277

Lung 0.512

4,116,4146,741,772

15,416,44085% 734 1,490 58 225

Ovary 0.512

3,522,1695,027,2496,962,983

92% 879 1,781 76 361

Brain 0.512

5,333,6086,988,354

12,433,09286% 1732 2,704 93 473

Breast 0.512

3,561,1906,728,231

15,849,29786% 756 2,033 113 447

Colon 0.512

2,739,6805,931,351

10,413,57092% 657 1,826 78 367

Heart 0.512

5,149,4865,929,0588,087,642

90% 983 2,496 50 335

Kidney 0.512

5,600,0648,488,442

12,285,99991% 1193 2,550 66 377

Liver 0.512

2,035,8477,048,78210,611,824

93% 387 1,304 37 183

Lymph Node

0.512

2,770,2364,822,8717,336,121

82% 399 1,511 52 303

Prostate 0.512

3,386,5183,498,4227,484,706

88% 113 786 21 123

Skeletal Muscle

0.512

3,838,3354,127,4646,645.,758

87% 727 1,528 40 237

Testis 0.512

4,580,4454,018,6073,858,634

77% 1086 3,308 123 660

Thyroid 0.512

2,905,8114,775,4694,326,014

92% 282 1,275 27 211

Average - 6,458,097 89% 647 1,807 66 332

Total 54 348,737,226 93% 9,254 1,2168 497 1,596


DDLD1 HEK293 HeLa MCF7

H3K4me1 Number of used reads 29,677,167 39,535,295 23,434,100 33,209,524

Number of MACS peaks 91,412 106,309 100,902 78,056



H3K27Ac Number of used reads 9,904,703 32,718,928 62,651,043 31,876,276


Pol II Number of used reads 14,471,858 9,926,213 139,395,594 35,905,171




CTD-PS2 Number of used reads 40,176,827 46,185,168 38,011,086 20,263,552


Rad21 Number of used reads 49,777,780 55,483,641 39,470,089 7,724,200


CTCF Number of used reads 40,743,100 29,032,630 24,818,196 46,715,322




A

B

C

RefSeq gene Promoter

NM PromoterNM

Alternative Promoter

NR PromoterNR

Alternative Promoter

81% 19% 77% 23%

RefSeq gene Terminator

NM TerminatorNM

AlternativeTerminator

NR TerminatorNR

AlternativeTerminator

40% 60% 49% 51%

DBTSS

DBTSSPromoter

DBTSSAlternative Promoter

DBTSSIntergenic

94% 67% 66%


our 5' cluster> 5ppm32,808

GIS-RNA-PET 5' cluster> 5ppm7,564

6,503


6,274 GIS-RNA-PET 3' cluster

> 5ppm7,624

D


PA-seq 3' cluster> 5ppm59,692

12,250

FE (top)

TSS tags in Hs

Random tags in Hs

TSS tags in Dm

Random tags in Dm

Gene model in HsGene model in Dm Sequence

alignment

Hs tag (1:exist)

Dm tag (1:exist)

Base aligned(1:yes) Tags

Number of genomic positions

in Hs

Number of genomic positionin Dm

Number of Hs tags Number of Dm tags

1 1 1TSS 532 513 6,616 887

PAS 25,548 8,523 5,553,380 135,869

1 1 0TSS 218 199 4,516 350

PAS 11,156 3,887 2,250,707 74,894

1 0 1TSS 394 23,235 5,574 0

PAS 14,990 258,723 1,112,303 0

1 0 0TSS 233 11,103 2,611 0

PAS 7,553 124,026 610,342 0

0 1 1TSS 198,911 10,288 0 25,724

PAS 181,694 2,597 0 49,156

0 1 0TSS 92,437 3,889 0 9,574

PAS 84,939 1,201 0 9,474

0 0 1TSS 199,543 365,344 0 0

PAS 177,148 129,537 0 0

0 0 0TSS 102,599 180,296 0 0

PAS 91,839 66,373 0 0

　　 total 　 1,189,734 1,189,734 9,546,049 305,928

SpeciesTotal number of valid tag pairs (TSS-Random)

Hs 348,708,022 pairs

Dm 3,465,945 pairs

Gene pairs in Homologene

(between Hs and Dm)

Aligned gene pairs (with E(1) < 1e-15)

Total aligned base

positions (including

gap)

Total identified base positions

Total mismatch

base positions (including

gap)

%identified

4,508 1,541 594,867 399,380 195,487 67.1%

E (bottom)


AAAAAA, 15%AAAAAAAA, 4%

AAAAAAAAAA, 2%

AAAAAAAAAAAA, 5%

other, 74%


0 5500 11000165002200027500330003850044000495000

1000

2000

3000

4000

5000

6000

First intron length (bp)

Num

ber o

f Ref

seq

NM

gen

es

8%

G

0

5

10

15

20

25

30

CGTA

GA GT GG GC

H

AATAAA, 39%

ATTAAA, 11%

AAAAAA, 8%ATAAAA, 2%

AAATAA, 2%AAAATA, 2%

ATAAAT, 1%TTTTTT, 5%

ATAAAG, 1%

TAAAAA, 1%

other, 28%

T T C G C T C T C T G C T C0

2000

4000

6000

8000

10000

12000

CGTA

GAPDH chr12: 6643680-6643697

I

%

tags

The last bases of the cap-oligo

Genome sequence5’-> 3’

First base of the TSSThe last bases of the cap-oligo

DDAH1 (dimethylarginine dimethylaminohydrolase 1)

85,880 kb85,960 kb86,040 kb

chr1:85,877,055-86,045,353

RefSeq gene model

TSS tags in DLD1


J

Distance from RefSeq NM_TSS (kbp)

Num

ber o

f Ref

Seq

NM

gen

es

-500

-450

-400

-350

-300

-250

-200

-150

-100 -5

0 0 500

500

1000

1500

15000

20000

Distance from RefSeq NM_PAS (kbp)

Num

ber o

f Ref

Seq

NM

gen

es

109,200 kb 109,250 kb 109,300 kb

ARMC2 (NM_032131; chr6:109,169,619-109,295,675 , strand : plus)

Distance from Refseq startTSS1 : +7 bp

Distance from Refseq startTSS2 : +68,519 bp

109,200 kb 109,250 kb 109,300 kb

Distance from Refseq endPAS1 : -94,217 bp

Distance from Refseq endPAS2 : -939 bp

0

(tag count)

100

0

(tag count)

100

ARMC2 (NM_032131; chr6:109,169,619-109,295,675 , strand : plus)

0100200300400500600

1200014000

12,819

19,091

-50

-40

-30

-20

-10 -5 0 5 10 20 30 40 50

0

200

400

1000

1200010,018

1,051N

umbe

r of R

efS

eq N

M g

enes

Distance from RefSeq NM_TSS (kbp)

-50

-40

-30

-20

-10 -5 0 5 10 20 30 40 50

0

200

400

600

800

1000

2000

9000

Num

ber o

f Ref

Seq

NM

gen

es

1,287

8,112

Distance from RefSeq NM_PAS (kbp)


ACTB(NM_001101)

2kb- +RT

full 0

(tag count)

50000

TSS PAS

1.5kb FAH(NM_000137)

full

- +

0

(tag count)

400

RT TSS PAS

3kb

- + - +

fullAlternativeTSC

ICAM1(NM_000201)

0

(tag count)200

RTTSS PAS

2kb- + - +

ACAD8(NM_014384)

fullAlternativePAC 0

(tag count)200

RTTSS PAS

2kb

full

- + - +

TRIP10(NM_004240)

0

(tag count)

30

Alternative TSC - PAC

RT

TSS PAS

2kb

Alternative TSC

Alternative PAC

- + - +

GTPBP1(NM_004286)

0

(tag count)500

RT TSS PAS

Z-score AP1-AT1 AP2-AT1

brain 2.45 2.34

heart 0.12 -0.04

colon 1.71 -0.31

Z-score AP1-AT1 AP2-AT1

testis -1.60 -2.49

colon -2.14 -3.34

heart 3.94 3.78

skeletalmuscle

2.52 3.55

KCNK1

brain

heart

colon

(tag count)

100

100

100

0

0

0

AP1 AT1AP2

APOBEC2(tag count)

1000

1000

1000

0

0

0

1000

0

testis

colon

heart

skeletal muscle

AP1 AT1 AT2


A

B C

++ +- --0

100

200

300

400

TATA

mot

if N

umbe

r of T

SC

pai

rs

CpG

isla

nd o

verla

pped

Num

ber o

f TS

C p

airs

++ +- --0

50 100 150 200 250 300 350 400

++ +- --0

100

200

300

400

Pol

y(A

)- a

dditi

on s

igna

l mot

if N

umbe

r of P

AC

pai

rs

　 DLD1 HEK293 Hela MCF7

Pol II GAPDH 30 (previous paper) 373.53 59.86 29.00GAPDH-359 56.93 7.45 773.00

H3K4me3 GAPDH 30 (previous paper) 7292.09 1222.78 272.58GAPDH-359 510.74 199.68 46.86

H3K27Ac ACTB 356 (previous paper) 2.87 192.73 113.99CDKN1A 3959 3.66 22.39 7.03

H3K27me3 HOXD13 -538 (previous paper) 7.24 37.44 5.21HOXD13 -925 6.55 43.31 7.35

H3K36me3 GAPDH 30.30 8.19 0.12 8.08ACTB 137.81 46.89 26.95 19.11

H3K4me1 GAPDH 2165 29.14 8.93 19.56 31.25CDKN1A 39.34 8.47 25.46 32.82

CTCFCTCF-1 N.S. 2.97 1.25 3.07CTCF-2 7.07 3.83 1.49 3.62CTCF-3 1.06 0.12 1.63 4.23

CTD-PS2GAPDH_3end 0.38 0.59 0.46 1.41

NM153339_3end 5.02 6.33 3.67 6.40NM004781_3end 1.20 2.15 0.41 3.08

Rad21 CSNP 8.18 31.62 1.85 8.61ERE2 18.23 7.24 4.46 16.19


A


B

HEK293

DLD1

MCF7

>5ppm

<5ppm

>5ppm

<5ppm

>5ppm

<5ppm

Signal intensity(Relative fold enrichment)

%0 100IP

/WC

E

fold

ave

rage

IP /

WC

E fo

ld a

vera

ge

-5 5 TSS PASTSS PAS-5 5 -5 5 -5 5 TSS PAS TSS PAS-5 5TSS TSS TSS TSS PAS

0 100%

H3K4me3 Pol II H3K27Ac CTD-PS2 Rad21CTCFH3K36me3 H3K27me3H3K4me1

IP/W

CE

fo

ld a

vera

geIP

/ W

CE

fold

ave

rage

IP/W

CE

fo

ld a

vera

geIP

/ W

CE

fold

ave

rage

C


HEK293

DLD1

MCF7

“Preferred” TSC-PACsin DLD1

“Preferred” TSC-PACsin other cell types

“Preferred” TSC-PACsin HEK293


“Preferred” TSC-PACsin MCF7


Signal intensity(Relative fold enrichment)

%0 100

-5 5 TSS PASTSS PAS-5 5 -5 5 -5 5 TSS PAS TSS PAS-5 5TSS TSS TSS TSS PAS

0 100%

H3K4me3 Pol II H3K27Ac CTD-PS2 Rad21CTCFH3K36me3 H3K27me3H3K4me1

IP/W

CE

fo

ld a

vera

geIP

/ W

CE

fold

ave

rage

IP/W

CE

fo

ld a

vera

geIP

/ W

CE

fold

ave

rage

IP/W

CE

fo

ld a

vera

geIP

/ W

CE

fold

ave

rage


Authentic model

Chromosome A

Possible inter-chromosomal fusion

Chromosome A

Chromosome B

Possible intra-chromosomal fusion

Chromosome A

>3M bp

A

B

C

dT Library Authentic model Inter-chromosomal Intra-chromosomal

TSC-PAC (>5ppm) TSC-PAC (>5ppm) TSC-PAC (>5ppm)

# Total Pair-Cluster44,902 4 1

5’ Gene5’ location

(chromosome, strand , TSS position)

3’ Gene3’ location

(chromosome, strand, PAS position)

DLD1 HEK293 HeLa MCF7 LC2AD

tag counts(ppm)

BCAS4 chr20 + 49,411,598 BCAS3 chr17 + 59,469,767 0 0 0 3213 0

MCTS1 chrX + 119,737,982 PSIMCT-1 chr20 + 30,135,753 190 213 282 157 168PIGY chr4 - 89,444,906 ICA1L chr2 - 203,637,882 5 7 0 0 9

BCAS4 chr20 + 49,411,598 REG4 chr1 - 120,336,729 0 0 0 23 0CCDC6 chr10 - 61,666,314 RET chr10 + 43,622,123 0 0 0 0 19


RT - NDUFA13 / YJEFN31.5kb

+

600

0

(tag count)TSS PAS TSS PAS

4000

VAMP8 / VAMP52kb

-

+

0

(tag count)

RT

TSS PAS TSS PAS

10000HLA-A / HLA-J

1.5kb -

+

0

(tag count)RT

TSS PAS

TSS PAS

30000

RPLP2 / CD1511.2kb -

+

0

(tag count)RT

TSS PAS TSS PAS

50HOXA4 / HOXA31.5kb-

+

0

(tag count)RT

TSS PAS TSS PAS


gap

Gene model

TSC-PAC Represent TSS Represent PAS

Assemble target

Tophat2 junction

Tophat2 mapped reads (TSS/PAS)

Tophat2 mapped reads (TSS/Random)

Assembled region

A


Supporting split tags(number <)

Known junction

Tophat unique Total

1 30,730 25,503 56,233

10 21,717 11,456 33,173

50 12,945 5,223 18,168

100 9,436 3,459 12,895

RefSeq junction

Tophat junction (from split tag)

Assembled model

B

Supporting split tags

(number <)

Constitutivedonor

Alternativedonor

(number of total combinations)

Constitutiveacceptor

Alternativeacceptor

(number of total combinations)

1 39,145 6,127 (17,088) 43,143 5,357 (13,090)

10 25,121 3,077 (8,052) 27,449 2,474 (5,724)

50 14,833 1,332 (3,335) 15,829 1,063 (2,339)

100 10,792 862 (2,103) 11,407 670 (1,488)


chr19:24,216,222-24,241,128RT

1kbp300

- +

0

(tag count)

chr5:179,894,258-179,910,338 200

- +

1kbp

0

(tag count)RT

chr11:2,454,029-2,465,78020

- +

1kbp

0

(tag count)RT

chr12:104,357,876-104,359,488 100

- +

1kbp

0

(tag count)RT

chr14:54,173,737-54,317,608400

- + 1kbp

0

(tag count)

RT

chr18:33,760,073-33,767,365- +

4001kbp

0

(tag count)RT

chr21:40,389,228-40,394,316- +

501kbp

0

(tag count)RT

　 Cell Gene　 Forward Primer Reverse Primer

LC2AD CCDC6 / RET GCAGCAAGAGAACAAGGTGC ACCATCCTAAGTTGCTGGGC

MCF7 BCAS4 / BCAS3 CCTCCTGATGCTGCTCGT AACCACGGTTTCCTTCACTG

Primers for validation analysis of the fusion gene (Fig. 5)

Supplementary Table1

Primers for validation analysis of the TSCs and PACs (Fig. S3)

Forward Primer Reverse PrimerACTB (NM_001101), full TTTTTAAGGTGTGCACTTTTATTCA AGCACAGAGCCTCGCCTTTFAH (NM_000137), full GGCGAGACTGTGGAGGAC CCTCAGAGCACAATGGCTTTAICAM1 (NM_000201), Alternative TSC – PAC ATTTTCCCAGGCCTTCTGAG CAGGTTGGCCAATGAGAAGTICAM1 (NM_000201), full CGCTGAGCTCCTCTGCTACT CAGGTTGGCCAATGAGAAGTACAD8 (NM_014384), TSC – Alternative PAC AGGCGTTCAGACTCTTAGCTG GGATCCCTGAGAACCACGTAACAD8 (NM_014384), full AGGCGTTCAGACTCTTAGCTG AAAGCCAAATTGGACGAGAGTRIP10 (NM_004240), Alternative TSC – Alternative PAC CTCCTTCTAGGAGCGCAGAG GAGTTCAGGCCGTCCATCTRIP10 (NM_004240), full CTCCTTCTAGGAGCGCAGAG CCGGTGTGACAGGTAGGACTGTPBP1 (NM_004286), Alternative TSC – PAC GTGGGCACTTCCTACAGTGG AAGAAGGTGGGGAGGACAGTGTPBP1 (NM_004286), TSC – Alternative PAC AAGTTATTAAAGATGGCGAC CTCAGTGTTGTCCCCGAAAC


Primers for validation analysis of the ChIP Seq of histone modifications (Fig. S5)

antibody 　 Region Forward Primer Reverse Primer

Pol II / H3K4me3 GAPDH 30 CTCTGCTCCTCCTGTTCGAC CTAGCCTCCCGGGTTTCTCTGAPDH-359 CAATTCCCCATCTCAGTCGT GCAGCAGGACACTAGGGAGT

H3K27Ac ACTB 356 TCTTGCCAATGGGGATCG CAGTTAGCGCCCAAAGGACCDKN1A 3959 AAAATCCAGTTGCTGCCAAG AGGAGAGGGAAAACGGAGAG

H3K27me3 HOXD13 -538 GGGCCTGTTTTCACATCATT TGTTTTTGCCACCTCTCCTCHOXD13 -925 CGTTCTCAGGGTCTTTCTGC CAACGCTCTTCGCCTCTACT

H3K36me3 GAPDH CTTCGTATGACTGGGGGTGT GGCCCAAGAGGTTGAATTTTACTB TTTGCACTTTCTGCATGTCC GGGGTGTTGAAGGTCTCAAA

H3K4me1 GAPDH 2165 CCCCTTCATACCCTCACGTA GACAAGCTTCCCGTTCTCAGCDKN1A AAAATCCAGTTGCTGCCAAG AGGAGAGGGAAAACGGAGAG

CTCFCTCF-1 TGGGAAAATTCCTCAAACCA CGCCTATAGACAACAAGTGCAACTCF-2 GGTCAGACACTCTGGCCTGT TTCAGCCATAGAGCTCCACACTCF-3 TTGCAGAGGTACAGGGCTTT TGAGACTGAGCAGAGCTCCA

CTD-PS2GAPDH_3end CGGCCTCTTTTGCTTTCTTA GGAGGACGTGGGAATCAGTANM153339_3end TGCCTGGTCTCCACAGTAGC TTAGGGCAGGTGGTCCTAGANM004781_3end TGTTCCCTTAACATCGCTGA CAGGAGCCAGTGGGTTACAT

Rad21 CSNP AACTTTCCCAGCCTCGTTCT GTCAGAGGTGGAGCTGGAAGERE2 GAGCTTCTCCCTAGCCCAGT CCAGATGAATTGCCCTCATT


Primers for validation analysis of the adjoining RefSeq transcripts (Fig. S7)

Forward Primer Reverse PrimerNDUFA13 / YJEFN3 CGTCAAAGGTGAAGCAGGA GGTGGGAGGGCTGTTTATTGVAMP8 / VAMP5 GGGCAAAGGTACCCCTTAGT AGCATGGCCAGTACAGCAGHLA-A / HLA-J CGGTCCCGGTTCTAAAGTC TGTCTTCCATTTATTTTGTCTCTCARPLP2 / CD151 CCGCGTGAGTGTGGTGAC TTTATTGAGCACCTGTATGTGGHOXA4 / HOXA3 TGTGGTTTGGACACTTCTGG AGATGGCCAATCTGCTGAAC


Primers for Validation analysis of the putative intergenic lncRNAs (Fig. S9)

Forward Primer Reverse Primerchr19:24,216,222-24,241,128 TGAAGAGGAGTGCATGGTTTC GAGGCAAGGAAATGGAGTCAchr5:179,894,258-179,910,338 GGGTTGTCCAAGAGCTTGTC TGTTGGCTCAGGGTAATGCTchr11:2,454,029-2,465,780 CACACAGTCTAGCCCCAGGT GTCTGCATCTCCCCTGCTTchr12:104,357,876-104,359,488 TTCAGTGTCCTCACCGACAA ACCAGAAGACGGGGACTAGGchr14:54,173,737-54,317,608 ATGCGCCTTGCCTACAAATA TGCATGGCATTTTCTCTGAAchr18:33,760,073-33,767,365 ATTTCGCTGTCTGACGTCCT GACCGGACTTTCACTTCTGGchr21:40,389,228-40,394,316 TGCCCCCATCACTTCTTAAA GACTGACATCGTGCCCTAGC


supplementary figure s1

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